Constrction Of CDNA Expression Library Of Ctenopharyngodon Idellus Induced By Poly I:C

With the increasing aquaculture scale and the reforming of aquaculture pattern, the fish diseases were increasingly serious, especially the large epidemic resulted from the viruses caused the large loss of the aquaculture, which was harmful to the aquaculture development. Interferon possessed the broad-spectrum antivirus functions and had the favorable effects in curing the human diseases caused by viruses. It was demonstrated that the recombinant interferon expressed in vitro was not harmful to the host and environment, so the using of the broad-spectrum antivirus genes have the expanded future in prevention and cure fish diseases.Since the first cDNA cloning experiment was occurred in mid-1970's, construction of the cDNA library became one of the basis methods to research functional genomics. The interesting gene can be screened from the cDNA library and then expressed. We can not only protect the cherished biological resource which was in severe danger, but also provide the probes for the construction of the molecular marker linkage maps. So the cDNA had the special advantages in the investigation of the expression and function of many genes in especial tissues, which facilitated to extensive applications in individual development, cellular differentiation, cellular cycle regulation, cell aging and death regulation and so on, and it was the basic library in research.The Ctenopharyngodon idellus cDNA library was constructed, and we could gain substantial immunogenes. It is very helpful to study the grass carp innate immunity and the related interferon system.Method: Many immunogenes was induced after injecting appropriate dose poly I:C to the healthy grass carps. mRNA was extracted, and the first stranded cDNA was synthesized by Oligo(dT) primers with the HA adaptor, then the double-stranded cDNA was amplified which was ligated to the EcoR I adaptor, so the cDNA library was established. The cDNA with the EcoR I adaptor was ligated to the plasmid vector digested with EcoR I restriction enzyme, and the grass carp cDNA library was constructed. The ZαcDNA fragment and the interferon cDNA fragment were screened from this library by the primers.Result: The average size of the constructed cDNA PCR library was about 1.5kb, which was agreed to the basic requirement, and the integrity was perfectible which was tested by immunogenes analysis. The size of the cloned cDNA fragment was 480bp, and this fragment was high homologous to the Ca-PKR Zαdomain through alignment. The identified IFN gene fragment, with the expected agreement, was about 400bp.It's proved that the library construction is fairly succeeded.